Aims: The main objective of this project is to develop a protein based therapy to enhance the endothelial barrier properties. Although acute lung injury (ALI) is a major cause of morbidity and mortality, no curative therapies are available. The main pathology in ALI is attributed to pulmonary edema, a direct result of endothelial (EC) barrier deterioration in the lung. In this project we will purify in their biologically active forms the barrier-protective proteins, Rac1, (-actinin and vinculin. Using our newly developed approach, we will attach these barrier-protective proteins to the cell-permeable peptide, TAT. The proteins will be given by mouse tail vein injection to determine their efficacy in protection against ALI. Procedures: We will use the Baculovirus Expression Vector System (BEVS) to develop the recombinant virus expressing the cDNA of the targeted proteins Rac1, vinculin and (-actinin as a His-tagged protein. This amplified virus will be used to infect the Sf9 insect cells to express each of these proteins. The expressed proteins will be purified from the cell lysates using Immobilized Metal Affinity Chromatography (IMAC). We will apply our newly developed approach to non-covalently complex the cell-permeable peptide, TAT, to His-tagged protein. We will expose cultured endothelial cells to the complexes. We will inject the complexes intravenously in mice. To determine uptake, we will image endothelial cells and lung microvessels by real-time fluorescence imaging. Significance: No therapy exists for ALI. Our research will establish for the first time, novel therapeutic modalities for curing this severe disease. Our approach for delivering therapeutic proteins is novel and potentially, superior to existing approaches. This research will determine preclinical feasibility of therapy that is potentially suitable for human application. PUBLIC HEALTH RELEVANCE: Although acute lung injury (ALI) is a major cause of morbidity and mortality, no curative therapies are available. Major pathology in ALI is attributable to pulmonary edema, which results from endothelial barrier deterioration in the lung. Hence, our aim is to develop therapy directed at endothelial barrier enhancement. We propose to introduce barrier-enhancing proteins in endothelial cells of lung microvessels. Feasibility of this approach is supported by our unpublished studies with purified focal adhesion kinase (FAKp). We have developed a new approach for chemically, non-covalently linking FAKp to the cell permeable peptide, TAT. The TAT-FAKp l Intraveously injected TAT-FAKp is taken up in lung microvessels. Intraveously injected FAKp protects against acid-induced ALI as determined by assays of lung endothelial permeability, extravascular lung water (EVLW) content and leukocyte and protein contents of the broncho alveolar lavage (BAL).We will apply our novel TAT linkage approach to develop intracellular delivery of three barrier-protective proteins, namely, vinculin, (-actinin and Rac1. Our approach for delivering therapeutic proteins is novel and potentially, superior to existing approaches. This research will determine preclinical feasibility of therapy that is potentially suitable for human application.